published_or_final_version / Biochemistry / Master / Master of Philosophy

published_or_final_version / Biochemistry / Master / Master of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Removal of the pineal gland from partially parathyroidectomized rats produces stereotyped violent seizures. Inasmuch as the neurotransmitter norepinephrine (NE) has been implicated in this experimental paradigm, the purpose of this study was to investigate the effect of specific alterations in catecholamine function on convulsions produced by pinealectomy (PinX). Additionally, the role of various pineal substances, sex differences and the caging paradigm in the convulsive response was studied. Male and female rats (grouped five per cage) were found to respond similarly to the convulsive stimulus of parathyroidectomy followed by PinX. Neither implants of melatonin nor ventricular injections of arginine vasotocin in isolated and grouped rats, respectively, produced consistent changes in convulsions from PinX. The method of caging the rats after PinX, however, dramatically influenced seizures. Isolated rats (one per cage) convulsed significantly later after PinX and did so less often than grouped (five per cage) controls. NE neurotransmission appears to play a strong role in influencing PinX-induced seizures. Augmenting NE function with desipramine suppressed seizures. Convulsions were enhanced by the (beta)-receptor antagonist timolol, while neonatal injections of the catecholamine neurotoxin 6-OHDA potentiated seizures so markedly that many rats died from just one convulsion. NE levels were significantly reduced in the telencephalons and increased in the brain stems of sham-pinealectomized rats which had also received neonatal 6-OHDA; telencephalic levels of DA were elevated by 6-OHDA. Both the proconvulsant effects of 6-OHDA and the alterations it produced in central catecholamine levels were prevented, for the most part, by pretreatment with DMI. Altering both NE and DA function with L-dihydroxyphenylalanine, (alpha)-methyl-p-tyrosine, FLA-63 or reserpine did not significantly affect PinX-induced seizures in isolated rats. NE appears to play a strong role in modulating PinX-induced seizures; however, a deficit in NE function per se does not seem to be the fundamental cause of the seizures since sham-pinealectomized rats having lowered NE and/or DA function did not convulse.

La resynchronisation biventriculaire est un traitement recommandé chez les patients avec dysfonction ventriculaire gauche (VG) sévère et QRS large. Si les résultats en termes de bénéfice clinique sont globalement positifs, toutes les études retrouvent un pourcentage non négligeable de patients non répondeurs à la thérapeutique. Dans ce travail, en couplant données expérimentales animales et études cliniques, nous avons 1) investigué l’impact de la resynchronisation biventriculaire chez des patients avec Tétralogie de Fallot opérée 2) évalué l’impact hémodynamique associé avec une stimulation VG multipoints et avec une stimulation VG endocardique 3) analysé l’intérêt de l’optimisation des paramètres de stimulation à l’effort. Nous avons mis en évidence que 1) la resynchronisation permet un bénéfice hémodynamique significatif sur un modèle animal de dysfonction ventriculaire droite et chez des patients avec Tétralogie de Fallot opérée 2) la stimulation multipoints et la stimulation endocardique VG permettent un bénéfice hémodynamique significatif sur des modèles animaux d’insuffisance cardiaque et chez des patients avec insuffisance cardiaque sévère 3) l’optimisation à l’effort des paramètres de programmation permet un bénéfice hémodynamique. Un capteur intégré dans la prothèse de stimulation pourrait permettre une optimisation automatique.L’ensemble de ces données permet d’espérer une optimisation de la réponse clinique et d’envisager de nouveaux champs d’application pour cette thérapeutique. / Cardiac resynchronization therapy is recommanded in patients with severe left ventricular (LV) dysfunction and wide QRS. Despite positive clinical results, a significant proportion of implanted patients do not demonstrate positive response to the therapy. Coupling experimental data and clinical studies, we have 1) investigated the impact of cardiac resynchronization in patients with repaired Tetralogy of Fallot 2) assessed the hemodynamic impact associated with multisite LV pacing and endocardial LV pacing 3) analyzed the impact of an exercise-optimization of the programmed parameters.We have demonstrated that 1) biventricular pacing is associated with a significant hemodynamic improvement in an animal model of right ventricular dysfunction and in patients with repaired Tetralogy of Fallot 2) multisite LV pacing and endocadial LV pacing are associated with significant hemodynamic improvement in animal models and in humans with severe heart failure 3) optimization during exercise of AV and VV delays induce acute hemodynamic improvement and could be automatically performed by an integrated hemodynimc sensor. Our data suggest a posible improvement in clinical response after cardiac resynchronization and a posible extension of the indications.

The general aim of the thesis was to deduce whether selected naturally occurring flavonoids (genistein, epicatechin gallate (EC), epigallocatechin gallate (EGCG), baicalin) attenuate various secondary insults that may cause death of ganglion cells in primary open angle glaucoma (POAG). An ischemic insult to the rat retina significantly causes the inner retina to degenerate indexed by changes of various antigens, proteins and mRNAs located to amacrine and ganglion cells. These changes are blunted in animals treated with genistein as has been shown for ECGC. Studies conducted on cells (RGC-5 cells) in culture showed that hydrogen peroxide, L-buthionine sulfoximine (BSO)/glutamate and serum deprivation (mimicking oxidative stress), rotenone, sodium azide (affecting mitochondria function in specific ways) and light (where the mitochondria are generally affected) all generated reactive oxygen species and caused death of RGC-5 cells. EGCG was able to attenuate cell death caused by hydrogen peroxide, sodium azide and rotenone. Only EC was able to attenuate BSO/glutamate-induced cell death, in addition to cell death caused by hydrogen peroxide and rotenone. Genistein had no positive effect on cell death in experiments carried out on RGC-5 cells. Exposure of RGC-5 cells to flavonoids showed that EC and EGCG increased the mRNA expression of endogenous antioxidants such as HO-l (heme oxygenase 1) and Nrf-2 (nuclear erythroid factor-z-related factor 2). Light insult, rotenone and sodium azide activate the p38 (protein kinase 38) pathway, while only light and rotenone activate the JNK (c-Jun amino-terminal kinase) pathway. Serum deprivation affects mitochondrial apoptotic proteins causing an increase in the ratio of Bax/Bcl2 (Bax: Bcl-2-associated X protein; Bcl-2: B-cell lymphoma 2). An insult of light to RGC-5 cells, unlike that induced by sodium azide, is inhibited by necrostatin-I and causes an activation of AlF (apoptosis-inducing factor) with alpha-fodrin being unaffected. These studies suggest that ganglion cell death caused by insults as may occur in POAG involves various cellular signaling pathways. The selected flavonoids have diverse actions in increasing cellular defense mechanisms, and in negating the effects of ischemia and specific types of oxidative stress. The results argue for the possible use of flavonoids in the treatment of POAG to slow down ganglion cell death.

The mismatching of alveolar ventilation and perfusion (VA/Q) is the major determinant of impaired gas exchange. The gold standard for analyzing VA/Q distribution is the multiple inert gas elimination technique (MIGET), conventionally based on gas chromatography (GC), and, although simple in principle, a technically demanding procedure limiting its use. A new technique based on micropore membrane inlet mass spectrometry (MMIMS) combined MIGET with mass spectrometry, simplifying the sample handling process, and potentially providing VA/Q distributions for a general clinical approach. The kinetics of volatile anesthetics are well known in patients with healthy lungs. The uptake and distribution of inhaled anesthetics have usually been modeled by physiologic models. However, these models have limitations, and they do not consider ventilation/perfusion matching. Respiratory diseases account for a large part of morbidity and mortality and are associated with pulmonary VA/Q mismatch that may affect uptake and elimination of volatile anesthetics. The objectives of the studies were firstly to investigate assessment of VA/Q mismatch by MMIMS and secondly to investigate the effects of asthma-like VA/Q mismatch on the kinetics of volatile anesthetics in an experimental porcine model. Anesthetized and mechanically ventilated piglets were studied. In study I, a direct comparison of MIGET by MMIMS with the conventional MIGET by GC in three animal models that covered a wide range of VA/Q distributions was preformed. The two methods agreed well, and parameters derived from both methods showed good agreement with externally measured references. In studies II–IV, a stable method of inducing and maintaining asthma-like VA/Q mismatch with methacholine (MCh) administration was established, and the effect of VA/Q mismatch on the pharmacokinetics of desflurane and isoflurane was investigated. The present model of bronchoconstriction demonstrates a delay in volatile anesthetic uptake and elimination, related to the heterogeneity of MCh-inhalation induced ventilation. The difference in solubility of volatile anesthetics has a significant influence on their uptake and elimination under VA/Q mismatch. The higher blood soluble isoflurane is affected to a lesser degree than the fairly insoluble desflurane.

La compréhension des mécanismes précis conduisant à la formation d'une plaque d'athérosclérose et à sa déstabilisation provoquant infarctus du myocarde ou accident vasculaire cérébral est fondamentale pour l'amélioration de la prévention, du dépistage et du traitement. Notre travail de thèse a porté sur l'analyse et le développement des modalités d'imagerie de la plaque d'athérosclérose afin d'améliorer notre capacité à suivre in vivo l'évolution de cette maladie. Nous proposons, après une introduction sur l'athérosclérose et la notion de plaque vulnérable, une revue des modèles animaux d'athérosclérose et l'intérêt du suivi en imagerie par résonance magnétique. Différentes modalités permettent le suivi en imagerie de la plaque d'athérosclérose. En pratique clinique, l'Imagerie par Résonance Magnétique (IRM) haute résolution est reconnue comme étant une modalité fiable et reproductible de caractérisation morphologique de la plaque carotidienne. Nous démontrons que l'injection de Gadolinium utilisée classiquement pour visualiser la lumière des vaisseaux permet également de préciser la caractérisation de la plaque carotidienne en donnant des informations sur la néovascularisation, la matrice extracellulaire et son statut inflammatoire. La caractérisation fonctionnelle de la plaque peut également être appréciée par l'injection de particules de fer en IRM ou par l'injection de radio-traceur comme le 18Ffluorodesoxyglucose en Tomographie par Emission de Positron (TEP). Le couplage de l'IRM et de la TEP de développement récent nous a permis de montrer une plus grande sensibilité de signal en TEP qu'en IRM avec particules de fer pour détecter les changements inflammatoires observés dans des plaques d'athérome d'aorte de lapin. Les applications actuelles de l'imagerie de l'athérosclérose sont nombreuses permettant de juger de l'efficacité thérapeutique d'un médicament ou d'identifier les patients à plus haut risque d'évènement cardiovasculaire / A better understanding of mechanisms leading to the formation of an atherosclerotic plaque and its destabilization causing myocardial infarction or stroke is fundamental to improve prevention, detection and treatment. Our work focused on development of imaging modalities of atherosclerotic plaque in order to improve our ability to monitor in vivo the progression of this disease. We propose, after an introduction to the concept of atherosclerosis and vulnerable plaque, a review of existing animal models of atherosclerosis and the interest of monitoring with magnetic resonance imaging. Different methods allow imaging of atherosclerotic plaque. In clinical practice, Magnetic Resonance Imaging (MRI) is recognized as a reliable and reproducible tool for morphological characterization of carotid plaque. We demonstrate that Gadolinium conventionally used to visualize the lumen of the vessels also allows a characterization of carotid plaque with information on neovascularization, extracellular matrix and inflammatory status. The functional characterization of the atherosclerotic plaque can be assessed with iron particles in MRI or with radiotracer such as 18Ffluorodeoxyglucose in positron emission tomography (PET). Combined PET-MR systems allowed us to show a better sensitivity of PET signal than USPIO-MR signal to detect early inflammatory changes in atherosclerotic plaque of rabbit aorta. We also provide some current clinical applications of atherosclerosis imaging (drug efficacy or identification of high risk patients)

INTRODUÇÃO: O modelo eslastase-induzido de aneurismas tem se destacado, nos últimos anos, porque simula as características geométricas dos aneurismas intracranianos humanos. A elastase destrói as fibras elásticas e dilata as artérias. A papaína é uma enzima que ainda não foi usada com esta finalidade. Os objetivos deste estudo foram determinar se a papaína produz aneurismas saculares em coelhos, e comparar suas características macroscópicas e histológicas com as dos aneurismas elastase-induzidos. MÉTODO: Dezoito coelhos brancos Nova Zelândia (1,9-3,2 kg) foram divididos em 3 grupos: I- elastase (n=7), II- papaína (n=8) e III- cirurgia controle (n=3). Os animais foram submetidos à exposição cirúrgica do pescoço, sendo que a artéria carótida comum direita foi usada como teste e a artéria carótida comum esquerda como controle. No 21° dia após a cirurgia, os animais foram sacrificados para retirada das artérias, tomada de suas medidas e análise histológica. Considerou-se formação de aneurisma quando a artéria teste dilatou em relação ao seu controle. RESULTADOS: Não houve aneurismas no grupo cirúrgico controle. Houve formação de aneurismas nos grupos elastase (71,4%) e papaína (100%). A diferença do diâmetro das artérias testes e seus respectivos controles não foi significativa (p= 0,15) entre os grupos elastase (média= 1,2 ± 0,4mm) e papaína (média= 2,1 ± 0,4mm), embora houvesse tendência deste último à maior dilatação . A histologia demonstrou que a papaína produziu maior tendência à lesão endotelial, à trombose (p = 0,01) e à inflamação parietal do que a elastase. A análise da fibrose intimal foi prejudicada em 50% dos casos do grupo papaína devido à trombose acentuada. Não houve diferença significativa entre os grupos quanto ao espessamento parietal (p=0,81) e ao grau de destruição das fibras elásticas (p= 0,009). CONCLUSÕES: A papaína produz aneurismas com tamanhos semelhantes aos da elastase, contudo a papaína provoca maior lesão endotelial, maior trombose e maior inflamação do que a elastase / INTRODUCTION: The elastase-induced model of experimental saccular aneurysms has been relevant in the last years because it mimics the size and geometric features of human intracranial aneurysms. Elastase destroys the arterys elastic fibers and produces arterial enlargement. Papain enzyme is also elastolytic but it had not been tested on saccular aneurysms creation yet. The purpose of this study was determine if papain produces saccular aneurysms in rabbits and to compare its gross and microscopic features are with the elastaseinduced aneurysms. METHODS: Eighteen New Zealand white rabbits (1.9 kg 3,2 kg) were separated in 3 groups: 1) sham (n=3) 0,9% saline solution; papain (n=8) 17-40 U; elastase (n=7) 6-8 U. The animals underwent surgical exposure of the neck; the right common carotid artery (RCCA) was used as the test and the left common carotid artery (LCCA) as the control. On the 21st day after surgery, animals were sacrificed for removal of the arteries, measurements and histological analysis. We determine formation of aneurysm to occur when the test artery dilated compared to the control. RESULTS: The sham group didnt develop aneurysms. There was aneurysm formation in the elastase (71,4%) and papain (100%) groups. The difference of the diameter of the tests and their respective controls is not significant (p=0,15) between elastase (average = 1,2 ± 0,4 mm) and papain (average = 2,1 ± 0,4mm) groups although there was tendency of this last one to produce larger aneurysms. the and to thrombosis (p = 0,01) and to parietal inflammation than the elastase. The analysis of the intimal fibrosis was not possible in 50% of papain cases due to pronounced thrombosis. There was no significant difference between the groups regarding the parietal thickening (p = 0,81) and the degree of destruction of the elastic fibers (0,009). CONCLUSION: Papain creates saccular aneurysms with similar dimensions to elastase-induced aneurysms. The microscopic results indicated papain destroys more endothelial cells, produces more thrombosis and more inflammatory process than elastase

Objetivo: Avaliar eletrofisiologicamente a função da retina do camundongo modelo de Alzheimer (3xTg-AD) comparando com seu controle (b6;129-PS1) em um estudo longitudinal com seis idades (2, 4, 6, 8, 10 e 12 meses). Métodos: Eletrorretinogramas (ERGs) foram registrados em 44 camundongos 3xTg-AD e em 23 controles, após administrada anestesia. Para o registro foi colocado um eletrodo de lente de contato sobre a córnea, um eletrodo de referência na cabeça e um terra na cauda. Em sessão de 30-40min de duração foram expostos ao seguinte protocolo de estimulação: 1) Adaptação ao escuro seguida de flashes nas intensidades: 0,003; 0,03; 0,3; 3 e 30 cd.s/m2; 2) Estimulação periódica (30 cd.s/m2) nas freqüências de 12, 18, e 30 Hz, sob luz de fundo (30 cd/m2). Resultados: Os ERGs mostraram dois tipos de respostas escotópicas tanto no grupo dos camundongos controles (b6;129- PS1) quanto nos modelos de Alzheimer. 13% dos camundongos controles e 72% dos modelos de AD apresentaram ERGs com potenciais oscilatórios presentes e tempo implícito da onda-b dentro da faixa esperada (45,31 ± 6,74 ms), enquanto no restante dos grupos, o ERG apresentou latência da onda-b muito aumentada (111,73 ± 22,56 ms) e potenciais oscilatórios ausentes. Devido a estes resultados, os grupos controle e experimental foram subdivididos em: b6;129 com OP, b6;129 sem OP; 3xTg-AD com OP e 3xTg-AD sem OP. Também foi incluído um grupo controle adicional constituído por 9 camundongos C57/B6. Comparando os cinco grupos, nenhuma diferença foi encontrada em relação à amplitude e à latência da onda-a. A amplitude da onda-b também foi semelhante para todos, ao contrário da latência para atingir o pico da onda-b dos grupos b6;129 sem OP e 3xTg-AD sem OP, que se apresentou duas vezes maior do que nos grupos com OP. As amplitudes dos cinco potenciais oscilatórios foram medidas individualmente e não mostraram diferenças entre os controles e os 3xTg-AD. Para o estímulo periódico, a amplitude do 1º harmônico dos grupos com OP mostrou clara diferença entre os grupos controle e o 3xTg-AD, tanto em 12 Hz como em 18 Hz. Os resultados dos dois grupos controle b6;129 e C57/B6 mantiveram-se muito próximos. Os grupos sem OP mantiveram-se sempre próximos a 10 V para as três freqüências de estimulação e mostraram atraso na diferença de fase média do 1º harmônico em 18 e 30 Hz, indicando maior lentidão de resposta, quando comparados aos primeiros. Conclusão: O camundongo 3xTg-AD e seu controle (b6;129) apresentam uma variante lenta e sem OPs do ERG escotópico em parte da população. Células bipolares, amácrinas e ganglionares podem estar alteradas nesses subgrupos (b6;129 sem OP e 3xTg-AD sem OP). Os grupos controle e 3xTg-AD com OPs diferiram quanto à amplitude de resposta à estimulação intermitente, diferença essa que implica em menor capacidade de processamento temporal para o modelo de AD. Sugerimos que as células bipolares de cones podem estar alteradas nos modelos de AD devido às amplitudes mais baixas dos 1os harmônicos desse grupo / Objective: To evaluate electrophysiologically the function of the retina of the Alzheimer model mouse (3xTg-AD) comparing it with its control (b6;129-PS1) in a longitudinal study at six ages (2, 4, 6, 8, 10 e 12 months) Methods: Electroretinograms (ERGs) were recorded in 44 anesthetized mice 3xTg-AD and in 23 controls, with a contact lens electrode placed on the cornea, a reference electrode on the head and a ground on the tail. During a 30-40min duration session the mice were exposed to the following stimulation protocol: 1) Scotopic response Dark adaptation followed by flashes at the following intensities: 0,003; 0,03; 0,3; 3 e 30 cd.s/m2; 2) Periodic stimulation (30 cd.s/m2) at the temporal frequencies of 12, 18, e 30 Hz, under background light (30 cd/m2). Results: The ERGs showed two types of scotopic responses, which ocurred in both the control mice (b6;129- PS1) and the Alzheimer´s models (3xTg-AD). 13% of the controls and 72% of the Alzheimer´s models mice presented ERGs with oscillatory potentials (OPs) and b-wave implicit times within the expected range (45,31 ± 6,74 ms), while for the other groups the ERG presented a very delayed b-wave latency (111,73 ± 22,56 ms) and absence of OPs. Given these results, the control and experimental groups were subdivided into: b6;129 with OPs, b6;129 without OPs; 3xTg-AD with OPs e 3xTg-AD without OPs. An additional control group with 9 mice C57/B6 was included. Comparing the five groups, no difference was found in a-wave amplitude and latency. The b-wave amplitude also did not differ among the groups, but the latency of the b-wave for the groups b6;129 without OPs and 3xTg-AD without OPs, was twice as long as in the groups with OPs. The amplitudes of the five OPs, measured individually, did not show differences between controls and 3xTg-AD groups. For the periodic stimulation the amplitude of the first harmonic of the Fourier transform of the groups with OPs showed a clear difference between the control and the 3xTg-AD groups, both for the 12 Hz and for the 18 Hz stimuli. The results of the two control groups (b6;129 and C57/B6) were very close. The groups without OPs had responses always close to 10 V for the three frequencies of stimulation and showed phase delay for the first harmonic, indicating response slowing, compared to the other groups. Conclusions: We found that a sub-group of both triple transgenic (3xTg-AD) and control mice (b6;129) manifest strikingly slow scotopic ERGs that lack OPs. We hypothesize that these response feature may reflect alterations in bipolar, amacrine and ganglion cells. The sub-group of triple transgenic and control mice that exhibited OPs differed in their response to flicker. Alzheimer model had significantly lower flicker-response amplitudes than the controls, suggesting impaired retinal temporal processing. We propose that the flicker results are consistent with alteration in cone bipolar cells in the Alzheimer model mice